Beta-cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D).1 Even prior to diagnosis, patients with impaired glucose tolerance (i.e., prediabetes) lack the first phase of glucose- stimulated insulin secretion and exhibit resistance to the amplifying effect of incretin hormones that normally prime the beta cell at meals.2 While deficient insulin secretion is now recognized as a prerequisite for both the onset and progression of T2D, the molecular pathways regulating this crucial physiologic response remain largely unknown. Moreover, no treatments are currently available to prevent the progressive nature of beta-cell decline characteristic of diabetes,3 and no small molecules exists that are FDA-approved to directly increase insulin secretion in a glucose-dependent manner, thereby avoiding the risk of hypoglycemia.4 To develop chemical probes of the molecular pathways regulating glucose-dependent insulin secretion and to identify small-molecule leads for glucose-dependent therapeutics, our proposed project wil employ a novel insulin secretion bioassay in the setting of near-stimulatory glucose conditions. Historically, a screen to identify modulators of insulin secretion has not been feasible, due to the lack of a suitable functional readout; the standard insulin ELISA is labor intensive, expensive, and limited to 96-well format. To overcome this bottleneck, we developed a high-throughput luminescent insulin secretion assay in which luciferase is targeted to the secretory vesicles of a beta cell an co-secreted with insulin upon stimulation. Luciferase serves as a close proxy for insulin, with enzyme activity responding appropriately to known secretagogues and inhibitors of insulin secretion, and in close correlation (r2 = 0.96) with insulin as measured by ELISA. Compounds found to increase luciferase secretion in near-stimulatory glucose concentrations will be counter- screened in the absence of glucose, to identify those exhibiting glucose-dependent effects. The activity of such compounds will be confirmed in a secondary assay using an insulin ELISA. Thereafter, the affected pathways within the beta cell will be explored using assays for ATP level and mitochondrial membrane potential. Lastly, top hits will be tested for their effect on dissociated human islets to confirm cross-species relevance. If successful, this phenotypic screen will identify new probes of pathways modulating beta-cell function in a glucose-dependent manner, improving our understanding of a causal disease mechanism for type 2 diabetes. In addition, this valuable toolbox of small molecules should prove useful to researchers exploring the response of the beta cell to known pathogenic insults, including ER stress, inflammation and glucolipotoxicity. Lastly, our screen may identify lead compounds for the development of therapeutics to safely treat diabetes without risk of hypoglycemia.